RECONSTRUCTION OF RBC CYTOSKELETAL ELEMENTS IN VITRO

我们打算研究细胞骨架蛋白在人红细胞凋亡中的作用， 血细胞（RBC）形成的结构元素能够借贷 完整的红细胞膜的弹性。 我们的做法是 在体外重组细胞骨架的可能元素， 纯化的细胞骨架蛋白。 我们将首先使用弹性 用振荡流变仪测量的模量，作为 体外 我们已经证明血影蛋白和肌动蛋白自发组装， 形成具有相当弹性模量的凝胶。 我们将调查 其他蛋白质如带4.1的作用以及 磷酸化和特异性离子结合血影蛋白，肌动蛋白和其他 细胞骨架成分 我们将确定的温度依赖性 我们的多蛋白凝胶的弹性模量。 作为下一步，我们建议开发一个2-D重建模型 利用磷脂的单层和Wilhelmy平衡。 我们希望 将纯化条带3插入到这样的单层中， 将血影蛋白-肌动蛋白-带4.1的元件转移到单层的溶液侧。 尽管这样的单层的绝对弹性和粘度是 由于溶剂的存在，表观弹性模量难以测量， 通过振荡Pt环确定的模量提供了一个很好的方法来跟踪 改变蛋白质组成和磷酸化的影响。 我们的目标是 为了定量地确定这种重构蛋白质的弹性， 络合物，并通过与全红的适当数据比较来确定 细胞的结构是否可能代表细胞骨架 建筑在体内。

We intend to investigate the role of cytoskeletal proteins of human red blood cell (RBC) in formation of structural elements capable of lending elastic properties to the intact RBC membrane. Our approach is to reconstitute in vitro possible elements of the cytoskeleton using highly purified cytoskeletal proteins. We will use, as a first step, the elastic modulus measured by an oscillating rheometer as a measure of elasticity in vitro. We have shown that spectrin and actin assemble spontaneously to form gels possessing respectable elastic modulus. We will investigate the role of other proteins such as band 4.1 as well as the role of phosphorylation and specific ion binding to spectrin, actin and other cytoskeletal components. We will determine the temperature dependence of the elastic modulus of our multiprotein gels. As the next step we propose to develop a 2-D model for reconstitution utilizing monolayers of phospholipids and a Wilhelmy balance. We hope to insert purified band 3 into such monolayer and attach reconstituted elements of spectrin-actin-band 4.1 to the solution side of the monolayer. Although the absolute elasticity and viscosity of such a monolayer is difficult to measure due to the presence of solvent, the apparent elastic modulus determined by oscillating Pt ring provides a good way to follow the effects of changing protein composition and phosphorylation. Our goal is to determine quantitatively the elasticity of such reconstructed protein complexes and determine by comparison to appropriate data for whole red cells whether the structures are likely to represent the cytoskeletal architecture in vivo.